Modified hpv e6 and e7 genes and proteins useful for vaccination

ABSTRACT

Described are DNA sequences encoding an E6 or E7 fusion protein of HPV, wherein said DNA sequences are characterized by a combination of the following features: original codons are exchanged by codons which lead to an enhanced translation in a mammalian cell, they contain a deletion resulting in the production of a truncated non-functional protein, and they encode a fusion partner which is a highly immunogenic polypeptide capable of enhancing the immunogenicity of the E6 or E7 protein in the mammalian host. Furthermore, the modified E6 or E7 protein encoded by said DNA sequences as well as expression vectors containing said DNA sequences are described as well as several uses of the these compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/472,724, filed Jan. 29, 2004, and issued Apr. 10, 2007 as U.S. Pat.No. 7,201,908, which was filed under 35 U.S.C. §371 and claims priorityof International Patent Application No. PCT/EP02/03271, filed Mar. 22,2002, which in turn claims priority of European Patent Application No.01107271, filed Mar. 23, 2001, all of which are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the invention

The present invention relates to DNA sequences encoding an E6 or E7fusion protein of HPV, wherein said DNA sequences are characterized by acombination of the following features: original codons are exchanged bycodons which lead to an enhanced translation in a mammalian cell, theycontain a deletion resulting in the production of a truncatednon-functional protein, and they encode a fusion partner which is ahighly immunogenic polypeptide capable of enhancing the immunogenicityof the E6- or E7-protein in the mammalian host. Furthermore, thisinvention relates to the modified E6- or E7-protein encoded by said DNAsequences as well as expression vectors containing said DNA sequences.Finally, the present invention relates to several uses of the abovecompounds, particularly as effective vaccines useful in treatment orprevention of an HPV infection or a neoplasm associated with HPVinfection.

2. Description of Related Art

Carcinoma of the uterine cervix (cervical cancer, CC) is the second mostcommon cancer in women worldwide and the first in developing countries.CC develops through premalignant intermediate stages of increasingseverity known as cervical intraepithelial neoplasia (CIN) grades 1-3,the latter leading to the development of invasive cancer in about 50% ofcases over a period of 1-2 decades. More than 11% of the global cancerincidence in women is due to human papillomavirus (HPV) infections.Infection with HPV types 16 and 18 has been associated with thedevelopment of CIN and CC, with HPV genotype 16 being the most prevalentviral type to infect the cervix. The E6 and E7 proteins encoded by theseHPV types are thought to be involved in the pathogenesis of CC byinducing abnormal cell proliferation. Expression of E6 and E7 isconsistently detected in tissue and tumor cells from HPV-associated CCs.Furthermore, the E6 and E7 genes from HPV types 16 and 18 are sufficientfor transformation of epithelial cells in culture (zur Hausen, Biochim.Biophys. Acta 1288(2) (1996): F55-78).

There is increasing evidence that the E6 and E7 proteins encoded by HPVtypes 16 and 18 may be effective immunological targets for tumorrejection by the host. Efforts are being made to develop effectivepreventive and therapeutic vaccines which may be useful in treatment andprevention of a neoplasm associated to HPV. The different strategiesemployed so far for inducing an immune responses to proteins of the HPVtypes 16 and 18 are: (a) Use of synthetic antigenic peptides, (b) Use ofrecombinant microorganisms (recombinant bacille Calmette-Guerin; BCG),(c) use of DNA vaccines using wild-type viral genes and (d) use ofVirus-like particles (VLPs).

However, unfortunately, the above strategies exhibit a variety ofdisadvantages which so far have hampered the development of a safe andefficient vaccine. As regards the use of synthetic antigenic peptides ithas to be stressed that the identification of HPV specific,immunoreactive peptides is very complex. It requires large numbers andquantities of peptides for vaccines to be effective and of a broadspectrum. Moreover, synthetic peptides do not contain posttranslationalmodifications (e.g., glycosylation, sulfation, phosphorylation) normallyfound in native proteins and therefore are not efficient enough asvaccines. The BCG based vaccine delivery systems expressing the L1 lateprotein of HPV 6b or the E7 early protein of HPV 16 have been used asimmunogens. However, the immune responses obtained with these systemswas even less than those elicited by protein/adjuvant vaccines and,thus, this system is considered unlikely to be useful as a singlecomponent vaccine strategy. As regards DNA vaccines it has been observedthat the expression of wild-type HPV genes is quite low, even if theyare expressed from strong promoters, such as that of the cytomegalovirus(CMV). As regards the use of Virus-like particles (VLPs) it has to bementioned that true VLPs are made of the L1 (capsid) protein of aspecific HPV type. Therefore, they may be only useful as prophylacticrather than as therapeutic vaccines, if ever. Pseudotyped VLPscontaining, for instance, epitopes of HPV-16 E7 have also been describedand may be useful as prophylactic and therapeutic vaccines. However, animportant limitation is that VLPs are produced in insect cells or inyeast. So far, no suitable production systems in mammalian cells havebeen established. Therefore critical epitopes depending onposttranslational modifications which take place in human cells are lostin these systems.

SUMMARY OF THE INVENTION

Therefore, it is the object of the present invention to provide a safeand effective vaccine, preferably a genetic vaccine, for the treatmentor prevention of an HPV infection or a neoplasm associated to HPV.

According to the invention this is achieved by the subject mattersdefined in the claims. The present invention provides DNA sequences forinducing immune response to the E6 and/or E7 proteins of oncogenic HPVin a host animal, preferably by administering vectors containing saidDNA sequences, e.g. plasmid vectors, herpes simplex virus type 1amplicon or recombinant Semliki forest virus vectors. Said DNA sequencesencode the HPV proteins as fusion proteins that are immunogenic but arenot capable of inducing cell transformation. The DNA sequences of theinvention are characterized by the following features:

-   (a) The DNA sequences of the HPV E6/E7 genes have been modified to    make their codon usage closer to that of human genes, (b) the genes    have been modified by deletion to make them non-functional, thereby    disabling their oncogenic capability (deletions are, preferably,    point mutations, because these lead to loss of potentially essential    epitopes), (c) the HPV genes have been fused to highly immunogenic    proteins to enhance their immunogenicity in the host (these fusions    are not expected to result in masking of HPV protein epitopes, since    the fragments fused are of sufficient length as to avoid this    problem), and, preferably, expression of the HPV genes is provided    by recombinant, replication-deficient HSV, SFV or high copy plasmid    vectors or combinations of these.

This approach offers a variety of advantages, namely:

-   (a) Higher expression levels of the HPV protein as a result of the    silent mutations introduced in the HPV genes to make their codon    usage closer to the human are obtained. This results in a more    efficient host response in immunization trials compared to the use    of wild-type HPV genes.-   (b) The HPV genes and proteins generated by the present invention    are expressed in human cells and, unlike proteins expressed in other    systems such as bacteria, yeast or insect cells, they contain    posttranslational modifications normally found in proteins expressed    in human cells. This is crucial for an adequate recognition of the    HPV proteins by the host immune system.-   (c) Since the HPV proteins are expressed fused to proteins known to    be highly immunogenic, they elicit stronger immune responses in the    host animal.-   (d) The HPV proteins are not cell-transforming neither in vitro nor    in the host animal because in no case are they expressed as    full-length polypeptides. The HPV fusion genes express incomplete    proteins, whose functions are impaired. In addition, the HPV    proteins are expressed as fusions to cytoplasmic proteins and    therefore they can not reach the nucleus where they exert their    functions.-   (e) The HPV proteins are, preferably, expressed tagged with a    specific sequence, which can be easily detected in Western blots and    by immunofluorescence with the help of commercially available    antibodies.-   (f) The combination of various viral vectors and of these with    plasmid vectors ensures a more efficient immunization, since it    prevents neutralization of the vector by immune reaction elicited in    a previous boost.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention relates to a DNA sequence encoding anE6 or E7 fusion protein of HPV, wherein said DNA sequence ischaracterized by a combination of the following features:

-   -   (a) at least 20% of the original codons are exchanged by codons        which lead to an enhanced translation in a mammalian cell;    -   (b) it contains a mutation resulting in the production of a        truncated non-functional protein; and    -   (c) it encodes a fusion partner which is a highly immunogenic        polypeptide capable of enhancing the immunogenicity of the E6 or        E7 protein in the mammalian host.

The expression “orignial codons” refers to the codons found in thecorresponding wildtype version of the HPV.

The expression “enhanced translation in a mammalian cell” refers to thegenes resulting from introduction of silent mutations in the HPVsequences, as described in the present invention, which create openreading frames consisting entirely of preferred human codons, and thuslead to enhanced expression of the proteins they encode in mammaliancells.

The term “mutation resulting in the production of a truncatednon-functional protein” refers to any mutation which leads to theproduction of a non-functional version of the protein. Preferably, sucha mutation leads to a truncated version of the protein. Examples ofappropriate mutations include a mutation, wherein at least one codon hasbeen deleted or a mutation leading to premature termination oftranslation. Such mutation is, e.g., the replacement of a codon encodinga particular amino acid by a stop codon, an insertion or deletion of oneor two nucleotides resulting in a frame shift mutation etc. The term“non-functional protein or gene” means that the mutant HPV genes andproteins of the present invention are “nontransforming neither in vitronor in vivo” meaning that the capability of the E6 or E7 genes andproteins to transform cells to a tumorigenic phenotype has beeneliminated as demonstrated by standard tests. The person skilled in theart can easily determine whether a particular mutation leads to an E6 orE7 gene or protein with the desired characteristics, i.e. which is“nontransforming” according to standard procedures. These include:

-   1) In vitro: Transformation assays of NIH 3T3 cells and primary    human keratinocytes. Transforming genes (oncogenes) have been    routinely identified by use of assays in which transformed foci    result from transfection of tumor or recombinant DNA into NIH 3T3    cells (Todaro et al., PNAS USA 51: 66-73, 1964; Jainchill et al., J.    Virol. 4: 549-553, 1969; Andersson et al., Cell 16: 63-75, 1979).    These cells are murine fibroblasts maintained as contact-inhibited,    non-tumorigenic cell lines. Transfer of DNA containing an acitivated    oncogene will occasionally give rise to foci of morphologically    altered cells that have tumorigenic properties.-   2) In vivo: Tumorigenicity tests are routinely performed in    immunodeficient mice by inoculation with mouse or human transformed    cells. Cells transfected to express HPV E6 and E7 genes and cell    lines derived from cervical carcinomas infected by HPV, such as HeLa    cells, have been shown to be tumorigenic (Lichy et al., Cell Growth    Differ. 3: 541-548, 1992; Stanbridge, Nature 260: 17-20, 1976).

In a preferred embodiment, the DNA sequence of the present inventionencodes the HPV E7 protein with the above described characteristics.

In a further preferred embodiment, at least 50% of the original codonsof the DNA sequence of the present invention are replaced by codonswhich lead to an enhanced translation in a mammalian cell; examples ofsuitable replacements are e.g., shown in FIGS. 1 and 2 SEQ ID NOs: 3 and1, respectively).

In a further preferred embodiment, the DNA sequence of the presentinvention contains a frame-shift point mutation leading to prematurestop of translation.

The person skilled in the art knows polypeptides or parts thereof whichare suitable as fusion partner for the E6 or E7 protein and which arehighly immunogenic in mammals, particularly in humans. Examples ofsuitable polypeptides include:

-   1) Hepatitis B virus small envolope protein (HBsAg-S). This protein    has the capacity to self-assemble with host-derived membranes to    form empty subviral particles, which are released into the lumen of    a pre-Golgi compartment and subsequently secreted (Ganem,    “Hepadnaviridae and their replication” p2703-37, in Fields, Knipe    and Howley (eds.), Fields Virology 3^(rd) ed., 1996,    Lippincott-Raven Publishers, Philadelphia). E6 or E7 can be fused to    the C-terminus of the protein which remains exposed on the surface    of the subviral particles.-   2) E2 glycoprotein of Semliki forest virus (SFV) . E2 is a spike    component of the SFV virion and a major antigen for neutralizing    antibodies (Schlesinger and Schlesinger, “Togaviridae: the viruses    and their replication” in Fields, Knipe and Howley (eds.), Fields    Virology 3^(rd) ed., 1996, Lippincott-Raven Publishers,    Philadelphia) . E6 or E7 can be fused to the N-terminus of the E2    protein that is exposed on the surface of the viral envelope or the    plasma membrane of E2-expressing cells.-   3) Human amyloid β-protein precursor (APP). APP is a transmembrane    protein with a large extracellular region and a small cytoplasmic    tail. It is normally cleaved by protease to yield a 40 amino acid    β-peptide (amyloid), which is found in the plaques of patients with    Alzheimer's disease, or a smaller fragment called p3, which may    associate with extracellular matrix (“Principles of neural Science”,    Kandel, Schwartz, and Jessell, (eds.) 3^(rd) ed., 1991, Elsevier,    New York) . E6 and E7 can be inserted into the extracellular part of    APP and are thought to be released together with the β-peptide or    the p3 fragment.-   4) Human chromogranin B (hCgB) . Although hCgB is a protein involved    in the regulated secretory pathway, it has been shown to be    constitutively secreted in cells without a regulated pathway, such    as HeLa cells, upon transfection (Kaether, and Gerdes, FEBS Letters    369: 267-271, 1995). E6 or E7 can be fused to the C-terminus of    hCgB.-   5) The bacterial B-galactosidase, known to be highly immunogenic    (Fijikawa et. al., Virology 204:789-793, 1994). E6 or E7 can be    fused to the N-or the C-terminus of the protein. As the fusion    product is a soluble non-membrane protein that may diffuse to the    nucleus, E6 or E7 is a deletion (inactive) mutant. Alternatively, a    signal peptide is added to the fusion which targets the product to    the cell surface.-   6) Fusion of the N-or C-terminal halves of E6 or E7 together and the    resulting chimeric polypeptide fused to any of the above proteins.

The present invention particularly, but not exclusively, relates to theE6 and E7 genes and proteins of the HPV-16 and HPV-18 genotypes. lt willbe, however, appreciated that the invention extends to variants of suchHPV genotypes and other HPV genotypes which may have oncogenic or otherpathologic potential.

In a preferred embodiment, the present invention relates to chimericgenes encoding a polyprotein containing E6 and E7 of HPV-16 and E6 andE7 of HPV-18, either complete or as deletion fragments comprising N- orC-terminal halves of such proteins, fused together and to thepolypeptides or parts thereof mentioned above. This allows immunizationagainst HPV16 and HPV18 using a single product as immunogen.

Persons skilled in the art will appreciate that the fusion of E6 and/orE7 to the proteins 1-4 of the above list abolishes the translocation ofthe former to the nucleus, thus interfering with their function.Further, secretion or surface exposure of the fusion proteins isintended to facilitate their recognition by the immune system.

In a particular preferred embodiment, the present invention relates to aDNA sequence wherein parts (a) and (b) comprise the coding region of theDNA sequence as depicted in FIG. 1 (SEQ ID NO: 3), 2 (SEQ ID NO: 1), 3(SEQ ID NO: 7), or 4 (SEQ ID NO: 5) including the Flag-tag or not. Evenmore preferred is an embodiment of the DNA sequences of the presentinvention, which comprises the coding region of the DNA sequence asdepicted in FIG. 5 including the Flag-tag or not.

Preferably, the mutant HPV E6 and E7 proteins encoding DNA sequences arepresent in a vector or expression vector. A person skilled in the art isfamiliar with examples thereof. In the case of an expression vector forE. coli these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b, T7 basedexpression vectors and pQE-8. For the expression in yeast, e.g. pY100and Ycpadl have to be mentioned while e.g. pKCR, pEFBOS, cDM8, pMSCND,and pCEV4 have to be indicated for the expression in animal cells. Thebaculovirus expression vector pAcSGHisNT-A is especially suitable forthe expression in insect cells. The DNA sequences of the presentinvention can also be contained in a recombinant virus containingappropriate expression cassettes. Suitable viruses that may be used inthe present invention include baculovirus, vaccinia, sindbis virus,SV40, Sendai virus, adenovirus, an AAV virus or a parvovirus, such asMVM or H-1. The vector may also be a retrovirus, such as MoMULV, MoMuLV,HaMuSV, MuMTV, RSV or GaLV. Particular preferred plasmids andrecombinant viruses are piRES-Neo2 (Clontech, Heidelberg, Deutschland),pTet-On (Clontech), pHSVPUC (Geller et al., PNAS USA 87 (1990),8950-8954), HSV amplicons and recombinant SFV vectors. For expression inmammals, the DNA sequences of the invention are operatively linked to asuitable promoter, e.g. a human cytomegalovirus “immediate earlypromoter” (pCMV), SV40 enhancer and early promoter, SRα (promoter(Takebe et al., Mol. Cell. Biol. 8: 466-472, 1988), Tet-On/Tet-Off geneexpression systems, immediate early E4/5 promoter of HSV-1 (Geller etal., PNAS USA 87: 8950-8954, 1990).

For generating E6 and E7 protein encoding DNA sequences carrying theabove discussed modifications and for constructing expression vectorswhich contain the DNA sequences according to the invention, it ispossible to use general methods known in the art. These methods includee.g. in vitro recombination techniques, synthetic methods and in vivorecombination methods as described in Sambrook et al., MolecularCloning, A Laboratory Manual, 2^(nd) edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., for example.

Furthermore, the present invention relates to host cells which containthe above described DNA sequences or vectors. These host cells includebacteria, yeast, insect and animal cells, preferably mammalian cells.The E. coli strains HB101, DH1, x1776, JM11, JM109, BL21, XL1Blue and SG13009, the yeast strain Saccharomyces cerevisiae, the insect cells sf9and the animal cells L, A9, 3T3, FM3A, BHK, human SW13, CHO, COS, Veroand HeLa are preferred. Methods of transforming these host cells, ofphenotypically selecting transformants and of expressing the DNAaccording to the invention by using the above described vectors areknown in the art.

The present invention also relates to an HPV E6 or E7 protein which isencoded by the above described DNA sequences. The HPV E6 or E7 proteinis provided as isolated, purified material, and therefore free of otherproteins. Such HPV proteins are, preferably, expressed in human cellsand, unlike proteins expressed in other systems such as bacteria, yeastor insect cells, they contain the posttranslational modificationsnormally found in the proteins expressed in human cells. This may be ofdecisive importance for an adequate recognition of the HPV proteins bythe host immune system.

Furthermore, the present invention relates to a method of producing theabove E6 or E7 protein, whereby, e.g., a host cell of the invention iscultivated under conditions allowing the synthesis of the protein andthe protein is subsequently isolated from the cultivated cells and/orthe culture medium. Isolation and purification of the recombinantlyproduced proteins may be carried out by conventional means includingpreparative chromatography and affinity and immunological separationsinvolving affinity chromatography with monoclonal or polyclonalantibodies.

The present invention also relates to a pharmaceutical compositioncomprising a DNA sequence or an expression vector of the invention or,alternatively, the HPV E6 or E7 protein encoded by said DNA sequence ina pharmaceutically acceptable carrier.

Finally, the present invention relates to various uses of the DNAsequences of the invention, expression vectors or HPV E6 or E7 proteins.Preferred uses are:

-   (a) Preparation of a vaccine for the prevention or treatment of a    HPV infection or a neoplasm associated with HPV infection.    Preferably, the vaccine is a genetic vaccine based on the DNA    sequences of the invention inserted into an appropriate vector under    the control of a suitable promoter, e.g. a vector or promoter as    described above. Such a vaccine can be used to stimulate humoral    and/or cellular immune response in subjects who may benefit from    such responses by protection against or treatment of possible    infections by HPV or by rejection of cells from tumors or lesions    which are infected by HPV and express viral proteins.-   (b) Production of polyclonal or monoclonal antibodies which might be    useful as therapeutic agents. Such antibodies can be generated    according to well known methods.-   (c) Detection of specific antibodies or cytotoxic T lymphocytes in    subjects infected by HPV, i.e. use in a diagnostic assay. Suitable    assay formats (RIA, ELISA etc.) are well known to the person skilled    in the art.-   (d) Generation of a transgenic mouse line using, e.g., the DNA    sequences of the invention under the control of a tetracycline    inducible promoter. Such mouse line might be useful to test vaccines    against HPV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HPV16 EE7T-sequence

The nucleotide sequence (SEQ ID NO: 3) and derived amino acid sequence(SEQ ID NO: 4)(single-letter code) of the mutagenized E7 gene of HPV-16(EE7T) is shown. Silent mutations which were introduced to create anopen reading frame of preferred human codons are denoted in bold. Thesequences encoding the hexa-His-tags and Flag-tags are underlined. Thestop codon is denoted by an asterisk.

FIG. 2: HPV16 EE6T-sequence

The nucleotide sequence (SEQ ID NO: 1) and derived amino acid sequence(SEQ ID NO: 2) (single-letter code) of the mutagenized E6 gene of HPV-16(EE6T) is shown. Silent mutations which were introduced to create theopen reading frame of preferred human codons are denoted in bold. Thesequences encoding the hexa-His-tags and Flag-tags are underlined. Thestop codon is denoted by an asterisk.

FIG. 3: HPV18 EE7T-sequence

The nucleotide sequence (SEQ ID NO: 7) and derived amino acid sequence(SEQ ID NO: 8) (single-letter code) of the mutagenized E7 gene of HPV-18(EE7T) is shown. Silent mutations which were introduced to create theopen reading frame of preferred human codons are denoted in bold. Thesequences encoding the hexa-His-tags and Flag-tags are underlined. Thestop codon is denoted by an asterisk.

FIG. 4: HPV18 EE6T-sequence

The nucleotide sequence (SEQ ID NO: 5) and derived amino acid sequence(SEQ ID NO: 6) (single-letter code) of the mutagenized E6 gene of HPV-18(EE6T) is shown. Silent mutations which were introduced to create theopen reading frame of preferred human codons are denoted in bold. Thesequences encoding the hexa-His-tags and Flag-tags are underlined. Thestop codon is denoted by an asterisk.

FIG. 5: HbsAg-EE7T Fusion Gene

The nucleotide sequence (SEQ ID NO: 9) and derived amino acid sequence(SEQ ID NO: 10) (single-letter code) of the HbsAg-EE7T fusion gene isshown. The fused fragments are separated by three dots. Silent mutationswhich were introduced into the E7 gene of HPV-16 (EE7T) to convert theopen reading frame of preferred human codons are denoted in bold. Thesequence encoding the Flag-tag is underlined. The stop codon is denotedby an asterisk.

FIG. 6: Confocal image analysis of expression of the HPV-16 E7 fusionproteins encoded by the mutant genes EE7T and E7T

Vero cells growing on coverslips were transfected with either plasmidpIRES-Neo2/EE7T or plRES-Neo2/E7T using the “FuGene” transfectionreagent (Roche, Basel Schweiz). The cells were incubated for 48 h, fixedwith 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 andstained by sequential incubations with anti-Flag M2 monoclonalantibodies (Sigma-Aldrich, Steinheim, Deutschland and goat anti-mouseantibodies conjugated to cy2 (Dianova, Hamburg, Deutschland).

FIG. 7 Western blot analysis of expression of the HPV-16 E7 fusionproteins encoded by the EE7T and E7T genes

HeLa cells were transfected as described in FIG. 6. After 24 h cellswere lysed in SDS buffer, proteins separated by PAGE (15%polyacrylamide), transferred to PVDF membranes Immobilion-P, Millipore,Eschborn, Deutschland) and hybridized with anti-Flag M2 monoclonalantibodies conjugated to horse-radish peroxidase, which activity wasdetected by ECL assay.

FIG. 8: Sequence of the synthetic EHBsAg-S-F gene and its translation

Silent mutations introduced to create an open reading frame of preferredhuman codons are denoted in uppercase characters. The sequence of theEco RV site is underlined. The amino acid sequence (SEQ ID NO: 27) isgiven below the nucleotide sequence (SEQ ID NO: 26) according to theone- letter code.

FIG. 9: Sequence of the EHBsAg-S-16EE7T fusion gene

The first codon of EE7T is underlined (SEQ ID NOs: 9 and 10).

FIG. 10: Immunofluorescence of Vero cells transfected withpIN-EHBsAg-S-EE7T

The cells were lysed 48 h after transfection, fixed with 4%paraformaldehyde, permeabilized with 0.1% (v/v) Triton X-100 in PBS andblocked. Immunodetection of the EHBsAg-S-EE7T fusion protein was carriedout using an anti- Flag M2 antibody (Sigma), followed by a Cy2 (green)conjugated anti-mouse antibody (Molecular Probes). The nuclei werecounterstained with propidium iodide.

FIG. 11: Western blot analysis of Vero cells transfected withpIN-EHBsAg-S-EE7T

The cells were lysed 48 h after transfection and equal amounts ofextract were loaded onto a 10% SDS-acrylamide gel. After transfer to aPVDF membrane and blocking, immunodetection was carried out withanti-Flag M2 antibody conjugated to horseradish peroxidase (Sigma).

FIG. 12: Immunogenicity of the EHBsAg-S-16EE7T fusion protein tested inBALB/c mice.

The present invention is explained by the examples.

EXAMPLE 1 Mutagenesis and Expression of the E7 Gene of HPV Type 16

The HPV-16 E7 gene was mutagenized in vitro to introduce 64 silentmutations which create an open reading frame comprised of preferredhuman codons. In addition, the mutant E7 genes were fused to ahexa-histidine-tag and a Flag-tag.

The mutant E7 genes were synthetically produced by sequential steps ofpolymerase-chain-reaction (PCR) using the following primers: (a) 5′-GGATCC AAG CTT GCC GTG ATC ATG (SEQ ID NO: 11) CAC GGC GAC ACC CCC ACC TTGCAC GAG TAC ATG TTG GAC TTG CAG CCC GAG ACC ACC GAC CTG TAC TGC TACGA-3′ (b) 5′-GTA GTG GGC GCG GTC GGG CTC GGC (SEQ ID NO: 12) CTG GCC GGCGGG GCC GTC GAT CTC GTC CTC CTC CTC GGA GCT GTC GTT CAA CTG C-3′ (c)5′-GCC CGA CCG CGC CCA CTA CAA CAT (SEQ ID NO: 13) CGT GAC CTT CTG CTGCAA GTG CGA CTC CAC CCT GCG CCT GTG CGT GCA GAG CAC-3′ (d) 5′-CCC GGGGAA TTC CTT AGG GCT TCT (SEQ ID NO: 14) GGC TGC AGA TGG GGC ACA CGA TGCCCA GGG TGC CCA TCA GCA GGT CCT CCA AGG TGC GGA TGT CCA CGT GG-3′ (e)5′-GAC CTG TAC TGC TAC GAG CAG TTG (SEQ ID NO: 15) AAC GAC AGC TCC GA-3′(f) 5′-AGG TGC GGA TGT CCA CGT GGG TGC (SEQ ID NO: 16) TCT GCA CGC A-3′(g) 5′-CAA GCT TGC TAG CAT GCA CCA CCA (SEQ ID NO: 17) CCA CCA CCA CGGCGA CAC CCC CAC CTT GCA CGA GTA-3′ (h) 5′-CAA GCT TGC TAG CAT GCA CCACCA (SEQ ID NO: 18) CCA CCA CCA CGA CGA GAT CGA CGG CCC CGC CGG CCA-3′(i) 5′-CGG ATC CGA ATT CTT ACT TGT CGT (SEQ ID NO: 19) CGT CGT CCT TGTAGT CGG GCT TCT GGC TGC AGA TGG GGC ACA-3′

In the first PCR step, primers (b) and (e) (PCR1) and primers (c) and(f) (PCR2) were used to generate the respective fragments by chainextension using no template. In a second step, the products of PCR1 andPCR2 were utilized to amplify a single fragment using no primers (PCR3).In a third step, the product of PCR3 was used as template to amplify acomplete E7 gene with primers (a) and (d) (PCR4).

In a final PCR step, the product of PCR4 (EE7) was utilized as templateto amplify the following : (1) by using primers (g) and (i) a completeE7 sequence fused to sequences encoding an hexa-His-tag (HHHHHH-epitope)(SEQ ID NO: 20) at its N-terminus, and a Flag-tag (DYKDDDDK-epitope)(SEQ ID NO: 21) at its C-terminus was synthesized (enhanced E7 withtags: EE7T); (2) by using primers (h) and (i) a truncated E7 (EE7TA1)lacking the first 35 residues, which contains His- and Flag-tags asdescribed above was synthesized.

The mutated tagged E7 genes were isolated from the PCR reaction mixturesby agarose gel electrophoresis, double digested with NheI and EcoRI andcloned into the multiple cloning site of the plasmid pIRES-Neo2(Clontech, Heidelberg, Deutschland) digested previously with the samerestriction enzymes. After transformation of DH5(bacteria, single cloneswere identified and sequenced. Clones with the correct sequence wereexpanded and used to purify the corresponding plasmids. As control, awild-type E7 gene and a truncated mutant lacking the first 35 residues,both tagged in the same way as the EE7T mutants described above, werecloned by PCR (E7T and E7TΔ1 genes), and subsequently inserted in theNhel and EcoRI sites of the pIRES-Neo2 plasmid.

The EE7 product from PCR4 was also cloned in pBluescript-vector(Stratagene, Amsterdam, Niederlande) and used for mutagenesis whichresulted in a double deletion mutant lacking residues 26-32 and 70-74.The EE7 product from PCR4 was used as template for amplification asfollows: (1) by using primers (g) and (i) an EE7T deletion mutantlacking residues 26-32 and 70-74 (EE7TΔ2,3), with His- and Flag-tags asabove was generated, (2) by using primers (h) and (i) a truncated EE7Tlacking the first 35 residues as well as residues 70-74 (EE7TΔ1,3), withHis- and Flag-tags as above was generated.

EXAMPLE 2 Expression of EE7T Fusion Genes in Mammalian Cells

The expression of the EE7T fusion genes described in Example 1, above,was tested in vitro by immunofluorescence and Western blot analysis ascompared to that of the E7T controls. The above plasmids were used fortransient transfection using eukaryotic cell lines of mouse (C-26),monkey (Vero 2-2), and human (HeLa) origin. The cell line Vero 2-2contains the HSV-1 IE2 (ICP27) gene and promoter. This line wasoriginally established by R.M. Sandri-Goldin et al. (Smith et al.,Virology 186 (1992), 74-86). At different times of expression the cellswere fixed with paraformaldehyde and processed for immunodetection orwere lysed in SDS loading buffer and analyzed by Western blot. In bothcases the E7 fusion proteins were detected with mouse monoclonalantibodies specific for the hexa-His (anti-His-tag Ab-1,Calbiochem-Novabiochem, Bad Soden, Deutschland) or the Flag epitopes(anti-Flag M2, Sigma-Alderich, Steinheim, Deutschland).

Image analysis of Immunofluorescence preparations showed expression ofthe mutant proteins in the nucleus of the transfected cells (FIG. 6).Western blots probed with monoclonal antibodies directed against theFlag epitope showed that expression of mutagenized E7 genes (EE7 and itsdeletion mutants described above) was at least two orders of magnitudehigher than that of equivalent E7 genes made of wild-type codons (VE7and its deletion mutants) (FIG. 7).

EXAMPLE 3 Cloning and Expression of E7/HBsAg Fusion Genes

In order to enhance the antigenic potential of E7, fusion proteins werecreated between tagged EE7 open reading frames and a gene encoding thesurface antigen of hepatitis B virus (HbsAg). The fusion gene wascreated by PCR cloning of the HbsAg and the EE7 genes. PlasmidpRc/CMV-HBs(S) (Aldevron, Fargo, USA) served as template to amplify theHbsAg gene using the primers 5′-CTC GAG GAT TGG GGA-3′ (SEQ ID NO: 22)and 5′-GAT ATC AAT GTA TAC CCA AAG A-3′ (SEQ ID NO: 23). The resultingfragment contains the full sequence of the HbsAg open reading frameexcept for the termination codon, which was replaced by an EcoRV site.The mutant EE7T genes were amplified using as template the full-lengthEE7 gene described in Example 1 and as primers for the 5′-end theoligonucleotides 5′-GAT ATC GAG GAG GAC GAG ATC GA-3′ (SEQ ID NO: 24) or5′-GAT ATC ATG CAC GGC GAC A-3′ (SEQ ID NO: 25) and for the 3′-end theoligonucleotide (i) described in Example 1. The EE7T genes amplified inthis way were cut with EcoRV and ligated to the 3′-end of the HbsAggenerated above to produce HbsAg-EE7T fusion genes expressing either thecomplete EE7 gene or the EE7TΔ1 or Δ deletion mutants.

The HbsAg-EE7T fusion genes were cloned into the polylinker of theplasmid pIRESNeo2 and used for transient transfection using eukaryoticcell lines of mouse (C-26; tumor library, DKFZ, Heidelberg, Germany),monkey (Vero 2-2), and human (HeLa) origin.

EXAMPLE 4

1. Transformation Studies of the Enhanced HPV Genes.

Experimental evidence has accumulated demonstrating that E6 and E7 fromHPV16 and HPV18 have tansforming potential. When expressed under thecontrol of strong heterologous promoters, these genes have been shown totransform established mouse cells (Kanda et al., J. Viol. 62: 610-613,1988; Vousden et al., Oncogene Res. 3:167-175, 1989) and to immortalizeprimary murine and human foreskin keratinocytes (Halbert et al., J.Virol. 65:473-478, 1991; Hudson et al., J. Virol. 64: 519-526, 1990;Sedman et al., J. Virol. 65:4860-4866).

The transforming potential of the enhanced genes of the presentinvention and of their derivatives (fusion proteins like that of FIG. 5and others in which the HPV gene has a deletion of at least 50%) wastested by standard methods using mouse NIH 3T3 cells and primary humankeratinocytes. Their wild type counterparts and empty plasmid vectorwere used as positive and negative controls, respectively.

The HPV enhanced genes and their fusion DNA constructs were subclonedinto the multiple cloning site of the plasmid pIRESNeo2 (Clontech,Heidelberg, Deutschland). The resulting plasmids were amplified in E.coli and purified on resin (Quiagen, Hilden, Deutschland), eluted,ethanol precipitated and resuspended in sterile, deionized water. DNAquanitity and purity was determined by spectrophotometric measurementsof absorbance at 260 and 280 nm and by agarose gel electrophoresis. NIH3T3 cells (ATCC, Manassas) were maintained on Dulbecco's modifiedEagle's medium supplemented with L-glutamine and 10% fetal calf serum

Transfection of NIH 3T3 cells with plasmid DNA was carried out usingFuGene™ 6 Transfection Reagent (Roche, Mannheim, Deutschland)essentially as described by the manufacturer. Cells seeded at 3×10 in a100 mm dish were transfected the following day with 3 μg of testplasmid. Each transfection was done in triplicate. After 48 h incubationat 37° C., transfected cells were removed by trypsinization and eitherassayed for colony formation in soft agar or subcultured into three 100mm dishes and incubated for further 24 h at 37° C. before selection wasperformed in medium containing Geneticin (Life Technologies, Karlsruhe,Deutschland) at a concentration of 500 μg/ml. For assays of colonyformation in soft agar, trypsinized cells were seeded into 0.4% agar ingrowth medium at 10⁵ cells per 60 mm dish and incubated at 37° C.Duplicate dishes were scored for colony formation after two weeks.Neomycin resistant colonies were selected by addition of Geneticin tosubconfluent cell monolayers, the cells were trypsinized and assayed forcolony formation in soft agar as described above.

Transfection of primary human keratinocytes with plasmid DNA was carriedout using FuGene™ 6 Transfection Reagent as above. Keratinocytes weregrown in KGM medium (KMK2 kit, Sigma-Aldrich, Steinheim, Deutschland) in30 mm dishes. Cells were transfected at passage 5 with 5 μg DNA. Afterapproaching confluence, the cultures were split at a ration of 1:2 andselection with 100 kg of Geneticin per ml was carried out.

All HPV enhanced fusion genes tested failed to produce foci of NIH 3T3cells in soft agar and to immortalize primary human keratinocytes.

2. Immunogenicity Sudies of the Enhanced HPV Genes.

The enhanced HPV genes were sub doned into the plasmid pHSVPUC (Gelleret al., PNAS USA87: 8950-8954, 1990) and the resulting recombinantconstructs used to generate amplicon HSV-1 vectors as describedelsewhere (Cid-Arregui, and Lim, in Cid-Arregui and Garcia (eds), “ViralVectors: Basic Science and Gene Therapy”, BioTechniques Books, EatonPublishing, Natick), and these used for immunization studies in BALB/cmice. Groups of five mice (8 weeks old, female) were used for eachimmunization experiment. On day 0, 10³-10⁴ virus particles in a 50 μlsuspension in saline serum were inoculated subcutaneously. At day 14, asecond dose of the formulation was applied in the same way. At day 28,the mice were bled.

Serum antibody responses to E6 and E7 were measured using plates coatedwith recombinant E6 or E7 protein using standard procedures. Sera werediluted in PBS pH 7.2 containing 1 mg/ml casein, 0.5% Tween 20, 0.002%alphazurine A.

After washing the plates, 0.1 ml/well of test serum at the appropriatedilution was added, and the plates incubated for 1 h at 38° C. To detectbound antibody, 0.1 ml of 0.1 μg/ml of horseradish peroxidase-labeledgoat anti-mouse IgG+IgM (H and L chain specific) in PBS pH 7.2supplemented as above was added. The plates were incubated for 1 h at20° C. and washed 6 times with PBS pH 7.2 with 0.5% Tween 20. Then 0.1ml of substrate TMB (3,3′,5,5′tetramethylbenzidine, Sigma-Aldrich,Steinheim, Deutschland) was added. Following 10 min of incubation at 20°C., the reaction was stopped by addition of 50 μl of 0.5 M H₂SO₄.Colorimetric measurements were performed in a vertical beamspectrophotometer at 450 nm.

All mice immunized with vectors expressing enhanced HPV E6 and E7 genesseparately or as fusion genes as described in the present inventionproduced a significant response following immunization which was clearlyhigher than that elicited by the non-enhanced controls.

EXAMPLE 5 Generation of a Synthetic EHBsAg-S-fusion Gene

The hepatitis B virus (HBV) small antigen (HBsAg-S) is an envelopeprotein with the capacity to self-assemble with cell-derived lipidmembranes into empty particles without the participation ofnucleocapsids. These subviral particles are produced as spherical orfilamentous forms of 22 nm in diameter, which bud into the lumen of apre-Golgi compartment and are subsequently secreted as cargo. It isbelieved that subviral particles induce a more effective immune responsethan denatured or soluble viral proteins. Furthermore, they can notreplicate and are noninfectious.

This example describes the development of recombinant HBsAg-S particlescontaining B- and T-cell epitopes of the E6 and/or E7 genes of oncogenicgenital HPV types fused to the C-terminus of HBsAg-S, and the humoralimmune response induced by these particles in mice.

1. Generation and expression of a synthetic HBsAg-S gene

A synthetic HBsAg-S gene was generated in vitro, which contains 155silent mutations that create an open reading frame entirely comprised ofpreferred human codons. Two extra codons (GATATC) were added justpreceding the stop codon which create an Eco RV restriction site thatallows for fusion of genes starting with an Eco RV site in frame attheir 5′ end. The resulting gene was named EHBsAg-S-F (Enhanced HBsAgfor Fusion). The synthetic EHBsAg-S-F gene was produced by successivesteps of polymerase-chain-reaction (PCR) using the followingoligonucleotides: (1) EH1 (forward) 5′ CTC GAG GAT TGG GGA CCC TGC GCT(SEQ ID NO: 28) GAA Cat gga gaa cat cac Ctc Cgg Ctt cct Ggg Ccc cct GctGgt gCT Gca ggc Cgg Ctt Ct 3′ (2) EH2 (anti-parallel) 5′ tCa gGg aGg tccacc aGg agt cCa (SEQ ID NO: 29) gGc tct gGg gGa tGg tCa gga tGc GGg tcaGca Gga aGa aGc cGg cct gCA Gca cCa gCa 3′ (3) EH3 (forward) 5′ tGg actcCt ggt gga cCt cCc tGa (SEQ ID NO: 30) aCt tCc tGg gCg gCa cCa ccg tgtgCc tGg gcc aGa aCt cCc agt ccc cCa cct cca aCc a 3′ (4) EH4(anti-parallel) 5′ aGc gGc gca gGc aca tcc agc gGt (SEQ ID NO: 31) aGccGg gGc aGg tGg gGg gGc aGg agg tGg gGg agt gGt tgg agg tGg ggg act gGgaGt t 3′ (5) EHS (forward) 5′ taC cgc tgg atg tgC ctg cgC cgC (SEQ IDNO: 32) ttC atc atc ttc ctG ttc atc ctg ctg ctG tgc ctG atc ttc Ctg CtggtG ctG ctg gac t 3′ (6) EH6 (anti-parallel) 5′ aGg gGc cGg tgc tgg tGgtGC Tgg (SEQ ID NO: 33) aGc cGg gGa tCa gGg gGc aCa cgg gca Gca tGc cCtgGt agt cca gCa gCa cca Gca Gga aga t 3′ (7) EH7 (forward) tcc AGC acCacc agc acC ggC ccC (SEQ ID NO: 34) tgc cgC acc tgc atg acC acC gcC caGggC acc tcC atg taC ccc tcc tgC tgc tgC a 3′ (8) EH8 (anti-parallel) 5′tGc cga aGg ccc agg aGC TGg gga (SEQ ID NO: 35) tgg gGa tGc agg tgc aGttGc cgt cGC TGg gCt tgg tGc agc aGc agg agg gGt aca tGg a 3′ (9) EH9(forward) 5′ atc ccC AGC tcc tgg gcC ttc ggC (SEQ ID NO: 36) aaG ttc ctGtgg gag tgg gcc AGC gcc cgC tt cAG Ctg gct Gag CCt Gct Ggt gcc Ctt Cgt3′ (10) EH10 (anti-parallel) 5′ acc aca tca tcc aGa tCa cGC TCa (SEQ IDNO: 37) gcc aCa cGg tgg ggC TCa gGc cCa cga acc act gCa cGa aGg gca cCagCa GGc tCa gcc a 3′ (11) EH11 (forward) 5′ tGA GCg tGa tCt gga tga tgtggt (SEQ ID NO: 38) aCt ggg gCc cCa gCc tgt aca gca tcC tga gCc cct tCCtG ccC ctg Ct 3′ (12) EH12 (anti-parallel) 5′ tta GAT ATC Gat gta Caccca Cag (SEQ ID NO: 39) Gca Gaa gaa Gat Ggg CaG cag Ggg CaG Gaa ggg GctcaG ga 3′

The synthetic EHBsAg-F gene was generated through four PCR steps asfollows:

In a first step, primers EH1 and EH2 (for PCR 1A), EH3 and EH4 (for PCR1B), EH5 and EH6 (for PCR 1C), EH7 and EH8 (for PCR 1D), EH9 and EH10(for PCR 1E), and primers EH11 and EH12 (for PCR 1F) were used togenerate fragments by chain extension using no template.

In a second step, the products of PCR 1A and 1B (for PCR 2A), 1C and 1D(for PCR 2B), and 1E and 1F (for PCR 2C) were utilized to amplify uniquefragments using primers EH1 and EH4 (PCR 2A), EH5 and EH8 (PCR 2B), andEH9 and EH12 (PCR 2C).

In a third step, the products of PCR 2A and 2B were used to amplify aunique fragment (PCR 3) without using primers.

In a final PCR step, the product of PCR 3 and 2C were mixed and used toamplify a unique fragment (PCR 4) using primers EH1 and EH12.

The resulting full length EHBsAg-S-F gene (715 base pairs in length,FIG. 8) (SEQ ID NO: 26) was isolated from the PCR reaction mixture byagarose gel electrophoresis, purified and cloned into a unique Eco RVsite in the polylinker of the pIRES-Neo2 plasmid (Clontech). Theresulting plasmid (pIN-EHBsAg-S-F) was purified from DH5x bacteria, andthe sequence of the EHBsAg-F gene verified by DNA sequencing usingprimers hybridizing upstream and downstream the polylinker.

Expression of the EHBsAg-S-F gene was tested by immunofluorescence andWestern blot analysis of transiently transfected cells. To this end, theplasmid pIN-EHBsAg-F was transfected into eukaryotic cell lines of mouse(C-26), monkey (Vero 2-2), and human (HeLa) origin using Effectene™(Qiagen) or FuGene™ (Roche). At different times of expression (24 and 48h) the cells were fixed with paraformaldehyde and processed forimmunofluorescence or lysed in SDS loading buffer and analyzed byWestern blot. In both cases the EHBsAg-S-F protein (SEQ ID NO: 27) wasdetected using mouse monoclonal antibodies specific for HBsAg-S(Aldevron).

Image analysis of Immunofluorescence preparations showed expression ofthe HBsAg proteins in the Golgi compartments of transfected cells.Western blots probed with monoclonal antibodies to HBsAg showedexpression levels about 5-10 times higher using the EHBsAg-S gene thanwhen the wild- type HBsAg-S gene was used for transfection.

2. Generation of Synthetic EHBsAg-S Fusion Genes

Fusions of the EHBsAg-S-F with synthetic HPV genes were generatedfollowing the strategy described below for the EE7T synthetic genedescribed in Example 1 and shown in FIG. 3. The HPV-16 EE7T genescontaining an Eco RV site at their 5′-end were amplified from thepIRESNeo2/EE7T plasmid by PCR using the following primers: 1) 167HLF5′:5′ GAT ATC ATG CAC GGC GAC A 3′ (SEQ ID NO: 40) 2) 167HSF5′: 5′ GAT ATCGAG GAG GAC GAG ATC (SEQ ID NO: 41) GA 3′ 3) 167HL3a: 5′ CGG ATC CGA ATTCTT ACT TGT (SEQ ID NO: 42) CGT CGT CGT CCT TGT AGT CGG GCT TCT GGC TGCAGA TGG GGC ACA 3′

The pair of primers 167HLF5′ and 167HL3a served to amplify a full lengthEE7T. The pair 167HSF5′ and 167HL3a was used to amplify a truncated EE7Tgene lacking the first 35 codons (EE7TA1). The resulting fragments weresequentially treated with T4-DNA polymerase, T4-polynucleotide kinase,restricted with Eco RV and purified using Qiaex II (Qiagen). Finally,the fragments were inserted, separately, into the plasmid pIN-EHBsAg-S-Fcut with Eco RV and Stu I. The sequence of the resulting fusion(plasmids pIN-EHBsAg-S-EE7T and pIN-EHBsAg-S-16EE7T, FIG. 9, andpIN-EHBsAg-S-16EE7T?1, respectively) was verified by sequencing.

Expression of the EHBsAg-S-16EE7T genes was tested by immunofluorescenceand Western blot analysis of transiently transfected cells. The plasmidspIN-EHBsAg-S-16EE7T and pIN-EHBsAg-S-16EE7T?1 were transfectedseparately into eukaryotic cell lines of mouse (C-26), monkey (Vero2-2), and human (HeLa) origin using Effectene™ (Qiagen) or FuGene™(Roche). At different times of expression (24 and 48 h) the cells werefixed with paraformaldehyde and processed for immunofluorescence (FIG.10) or lysed in SDS loading buffer and analyzed by Western blot. In bothcases the EHBsAg-S-16EE7T proteins were detected using mouse monoclonalantibodies specific for HBsAg-S (Aldevron) and anti-flag antibodies (M2mAb, Sigma) (FIG. 11).

3. Immunization of Mice with Synthetic EHBsAg-S-16EE7T Fusion Genes

Immunogenicity of the EHBsAg-S-16EE7T fusion protein was tested inBALB/c mice. On day 0, eight groups of three mice (10-12 weeks old,females) were inoculated with 10⁴ infectious units of herpes simplexamplicon expressing EHBsAg-S-16EE7T in 40 μl of buffer TN (50 mMTris-HCl pH7.4, 100 mM NaCl, 0.5 mM EDTA) either subcutaneously (dorsal,close to the head), intramuscularly (Tibialis anterior muscle) or bothsubcutaneously and intramuscularly. A second dose was administered toall groups on day 15.

All mice were bled at days 15 and 25. Serum antibody responses toEHBsAg-S-16EE7T were measured by EIA. Nunc 96-multiwell plates werecoated with recombinant HBsAg-S protein by incubating 0.1 ml/well for 2h at 37° C. of a 10 μg/ml in 4M urea in 50 mM carbonate buffer pH 9.5.The buffer was aspirated and the plates incubated at 37° C. for 1 h with0.2 ml/well of 1 mg/ml of casein in PBS pH 7.2. The plates were thenwashed six times with PBS pH 7.2, 0.5% (v/v) Tween 20. Test sera,diluted in PBS pH 7.2, 0.5% (v/v) Tween 20, 1 mg/ml of casein, wereadded and the plates incubated for 1 h at 37° C. The plates were thenwashed six times with PBS pH 7.2, 0.5% (v/v) Tween 20. Bound antibodywas detected by adding 0.1 ml/well of 0.1 μg/ml of horseradishperoxidase labelled goat anti-mouse IgG+IgM in PBS pH 7.2, 0.5% (v/v)Tween 20, 1 mg/ml of casein. The plates were incubated for 1 h at 20°C., washed six times with PBS pH 7.2, 0.5% (v/v) Tween 20,and incubatedfor 10 min with 0.1 ml of enzyme substrate (3,3′,5,5′-tetramethylbenzidine/H₂O₂). The reaction was stopped by addition of50 up of 0.5 M H₂SO₄. Color was measured at 450 nm in a plate reader(FIG. 12).

1. An E6 or E7 protein which is encoded by a DNA sequence encoding an E6or E7 fusion protein of HPV, wherein said DNA sequence is characterizedby a combination of the following features: (a) at least 20% of theoriginal codons are exchanged by codons which lead to an enhancedtranslation in a mammalian cell; (b) it contains a mutation resulting inthe production of a truncated non-functional protein; and (c) it encodesa fusion partner which is a highly immunogenic polypeptide capable ofenhancing the immunogenicity of the E6 or E7 protein in the mammalianhost.
 2. Use of an E6 or E7 protein according to claim 1 for theproduction of a polyclonal or monoclonal antibody.
 3. Use of an E6 or E7protein according to claim 1 in an assay for the detection of specificantibodies or cytotoxic T lymphocytes in subjects infected by HPV.
 4. Apharmaceutical composition comprising the E6 or E7 protein according toclaim
 1. 5. A method of determining whether a subject is infected withHBV, said method comprising detecting specific antibodies or cytotoxic Tlymphocytes in a biological sample from said subject using a diagnosticassay comprising the E6 or E7 protein according to claim
 1. 6. Themethod of claim 5, wherein said diagnostic assay comprises a RIA assay.7. The method of claim 5, wherein said diagnostic assay comprises anELISA assay.